During ischemia, myocardial fatty acid metabolism ceases, rapidly depleting the other primary fuel, glucose. No technique has existed to continuously monitor myocardial glucose. Needle-tip enzymatic glucose biosensors have been developed for subcutaneous use in diabetic management. To study the utility of these sensors for real-time myocardial glucose monitoring in clinically relevant applications, 40 kg Yorkshire swine were cannulated for cardiopulmonary bypass. Biosensors were placed in the left anterior descending artery distribution (LAD) and posterior descending artery distribution (PD), and a third in the liver. Selective ischemia was induced by ligation of the LAD artery. Glucose levels were monitored during ischemia and reperfusion in the setting of cardioplegic arrest (n = 7) and in the normal beating heart (n = 14). In the normal beating heart, glucose levels fall to 6.5% +/- 7.4% baseline at 1 minute and 29.0% +/- 23.0% at 5 minutes of ischemia. In both arrested and beating heart scenarios, biosensors show distinct metabolic states in specific regions of the heart and liver. Biosensors can track regional glucose metabolism in the beating and arrested heart. This novel application of these sensors allows real-time determination of myocardial glucose levels to guide cardioplegia administration and monitor ischemic states for clinical and experimental use.